RESEARCH

Mosaic RNA Phage VLPs Carrying Domain III of the West NileVirus E Protein

Indulis Cielens • Ludmila Jackevica •

Arnis Strods • Andris Kazaks • Velta Ose •

Janis Bogans • Paul Pumpens • Regina Renhofa

� Springer Science+Business Media New York 2014

Abstract The virus-neutralising domain III (DIII) of the

West Nile virus glycoprotein E was exposed on the surface

of RNA phage AP205 virus-like particles (VLPs) in mosaic

form. For this purpose, a 111 amino acid sequence of DIII

was added via amber or opal termination codons to the

C-terminus of the AP205 coat protein, and mosaic AP205-

DIII VLPs were generated by cultivation in amber- or opal-

suppressing Escherichia coli strains. After extensive puri-

fication to 95 % homogeneity, mosaic AP205-DIII VLPs

retained up to 11–16 % monomers carrying DIII domains.

The DIII domains appeared on the VLP surface because

they were fully accessible to anti-DIII antibodies. Immu-

nisation of BALB/c mice with AP205-DIII VLPs resulted

in the induction of specific anti-DIII antibodies, of which

the level was comparable to that of the anti-AP205 anti-

bodies generated against the VLP carrier. The AP205-DIII-

induced anti-DIII response was represented by a significant

fraction of IgG2 isotype antibodies, in contrast to parallel

immunisation with the DIII oligopeptide, which failed to

induce IgG2 isotype antibodies. Formulation of AP-205-

DIII VLPs in alum adjuvant stimulated the level of the anti-

DIII response, but did not alter the fraction of IgG2 isotype

antibodies. Mosaic AP205-DIII VLPs could be regarded as

a promising prototype of a putative West Nile vaccine.

Keywords West Nile virus E glycoprotein � Domain

DIII � RNA phage AP205 � Mosaic � Virus-like particles

Introduction

West Nile virus (WNV) is a neurotropic, single-stranded

and positive-sense RNA flavivirus that is transmitted to

humans through the bite of an infected mosquito and has

emerged globally as a significant cause of viral encephalitis

(for recent reviews see [1, 2]). In the absence of a specific

anti-viral treatment, the development of a safe and an

efficient prophylactic vaccine against WNV is necessary.

Currently, most WNV vaccine candidates are live,

attenuated viral vaccines based on chimeric viruses that

incorporate the pre-membrane (prM) and E glycoproteins

of the WNV envelope into the following viral vectors:

yellow fever [3–6], fowlpox and canarypox [7], measles [8]

and the modified vaccinia virus Ankara (MVA) strain of

vaccinia virus [9]. The generation and preclinical efficacy

of a hydrogen peroxide-inactivated WNV vaccine has been

described [10]. A DNA vaccine encoding the prM and E

proteins have been evaluated in healthy adults [11].

Recombinant subunit vaccine candidates are based on

the structural WNV glycoprotein E because the protein

may elicit a major neutralising antibody response [12, 13].

The recombinant E protein was purified from Escherichia

coli and functioned as an efficient WNV vaccine in mice

[14]. The WN-80E subunit vaccine, which is produced in a

Drosophila melanogaster expression system, consists of

the recombinant E protein truncated at the C-terminal end

but contains 80 % of its N-terminal amino acids (aa) [15–

17]. Recently, recombinant baculoviruses expressing WNV

E protein, as well as prM protein, were constructed and

tested successfully in mice [18, 19].

Within the E glycoprotein, domain III (DIII) is the region

that is exposed on the viral surface [20] and is implicated in

receptor binding [21]. DIII is a target of the most WNV

neutralising antibodies [22–26], and passive transfer of DIII-

I. Cielens � L. Jackevica � A. Strods � A. Kazaks � V. Ose �J. Bogans � P. Pumpens (&) � R. Renhofa (&)

Latvian Biomedical Research and Study Centre, Ratsupites

Street 1, Riga 1067, Latvia

e-mail: paul@biomed.lu.lv

R. Renhofa

e-mail: regina@biomed.lu.lv

123

Mol Biotechnol

DOI 10.1007/s12033-014-9743-3

specific antibodies may protect mice from WNV challenge

[27]. Subunit vaccines based on recombinantly expressed

DIII have been tested in animal models and have proven

effective in protecting against WNV infection [28–32].

The immunogenicity of DIII was strongly enhanced by

chemical conjugation to virus-like particles (VLPs) of the

bacteriophage AP205 [33], in accordance with the general

acceptance of VLPs as highly ordered carriers for foreign

epitopes (for recent reviews see [34, 35]).

In the present study, construction of a novel type of

putative VLP-based DIII vaccine is described. RNA bac-

teriophage AP205 VLPs [36] are used to expose the DIII

sequence on the mosaic VLPs. In contrast to the previous

chemically conjugated vaccine [33], mosaic AP205 VLPs

are generated by genetic fusion of the DIII sequence to the

C-terminus of the AP205 coat protein (CP) via the termi-

nation codons UAG and UGA under codon-suppression

conditions. Uniform fusions of the DIII sequence, without

any read-through termination codons, to AP205 CP as well

as to the CP of a similar RNA bacteriophage, GA, do not

lead to self-assembly and formation of VLPs. Direct

expression of the DIII sequence is used as a source of the

highly purified recombinant DIII protein. Overall, we

present efficient production and purification of mosaic

AP205-DIII VLPs in E. coli cells and show the ability of

mosaic VLPs to induce specific anti-DIII antibodies in

mice.

Materials and Methods

Bacterial Strains

Escherichia coli strain RR1 [F- rB- mB- leuB6 proA2 thi-1

araC14 lacY1 galK2 xyl-5 mtl-1 rpsL20 (Strr) glnV44 D(mcrC-mrr)] was used for the cloning and selection of

recombinant plasmids. E. coli C2566 was used for the direct

expression of the DIII gene. E. coli JM109 was used to

express the fused AP205-DIII and GA-DIII genes. For the

expression of mosaic AP205-am-DIII and AP205-op-DIII

VLPs, the amber suppressor E. coli JM109 (pISM579) and

the opal suppressor E. coli JM109 (pISM3001) carrying

resident suppressor tRNA genes were used. The plasmid

pISM3001 [37] was a kind gift from Dr. F.C. Minion (USA),

while strain MY579 harbouring amber suppressor tRNA was

obtained from Dr. M. Yarus (USA). The plasmid pISM579

encoding tRNA for amber (UAG) codon suppression was

constructed on the basis of the plasmid pISM3001, where the

opal (UGA) suppressor tRNA encoding gene trpT176 was

replaced with analogous DNA sequence encoding the Hirsh

amber suppressor tRNA. The latter was isolated from plas-

mid pBE621 [38] that encodes the trpT178 derivative,

which, in addition to Hirsh mutation G24 ? A, also contains

mutations U33 ? G and C35 ? U. Because the respective

tRNA genes are flanked by EcoRI sites, the substitution was

carried out by partial EcoRI cleavage and religation and then

confirmed by sequencing.

Construction of the WNV Protein E Domain DIII-

Expressing Plasmids

The construction map of the WNV protein E domain DIII-

expressing plasmids is shown in Fig. 1. To construct the

AP205-DIII expression units, a set of cloning vectors

(Fig. 1, on the left) was generated on the basis of a

pAP283-58 plasmid that expresses the CP gene of the RNA

bacteriophage AP205 under the control of the E. coli

tryptophan operon promoter Ptrp [36]. The following oli-

gonucleotides were used as PCR primers to insert the

sequences encoding the linker aa residues together with the

appropriate cloning sites and the suppression codons

(underlined) at the C-terminus of the AP205 CP gene:

50-TGTCTAGAATTTTCTGCGCACCCATCCCGG-30;50-TGATGCATCCTCCGGATCCAGCAGTAGTATC

AGACGATAC-30;50-TACCATGGCAAATAAGCCAATGCAACCG-30;50-GTAAGCTTAGATGCATTATCCGGATCCCTA

AGCAGTAGTATCAGACGATACG-30;50-GTAAGCTTAGATGCATTATCCGGATCCTCA

AGCAGTAGTATCAGACGATACG-30.

The WNV protein E DIII fragment encompassing aa

residues 296–406 was PCR-amplified from the plasmid

pTrcHis2-WNVclone F101 New York strain 385–399

(kindly supplied by B. E. E. Martina, Erasmus MC, Rot-

terdam) and cloned into the previously constructed vectors

at the appropriate restriction sites (Fig. 1, on the right).

The following oligonucleotides were used as cloning

primers for the PCR:

50-CATCCGGACAGTTGAAGGGAACAAC-30;50-GTATGCATTTGCCAATGCTGCTTCC-30;50-CATCCGGACAGTTGAAGGGAACAAC-30;50-GTAAGCTTATTTGCCAATGCTGCTTCC-30

To construct the GA-DIII expression units, a pGA

355-24 plasmid expressing the CP gene of the RNA bac-

teriophage GA under control of the Ptrp [39, 40] was

supplied with the appropriate linker at the C-terminus and

used as a vector for cloning of the DIII sequence amplified

with the following primers:

50-CATCCGGACAGTTGAAGGGAACAAC-30 and

50-GTAAGCTTATTTGCCAATGCTGCTTCC-30

For direct expression, the DIII sequence was amplified

by the primers

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50-TACCATGGGCCAGTTGAAGGGAACAACCTAT

GG-30 and

50-ATGAAGCTTATTTGCCAATGCTGCTTCC-30

and inserted at the restriction sites NcoI and HindIII in the

multicloning region of the plasmid pET-28? under control

of the IPTG-inducible T7 promoter. The plasmid structures

were confirmed by sequencing.

Expression and Purification of AP205- and GA-DIII

Derivatives

Escherichia coli JM109 cells were transformed with the

pAP205-DIII and pGA-DIII plasmids. E. coli JM109

strains carrying resident suppressor tRNA plasmids were

used for the expression of AP205-DIII mosaics. E. coli

JM109 with a resident amber suppression tRNA gene

(pISM579) was transformed with the pAP205-am-DIII

plasmid. E. coli JM109 with a resident opal suppression

tRNA gene (pISM3001) was transformed with the

pAP205-op-DIII plasmid. Single colonies were suspended

in tubes containing 5 mL of LB medium with 50 lg/mL of

ampicillin and 10 lg/mL of chloramphenicol and grown

without shaking at 37 �C for 16 h. The prepared inoculum

was diluted tenfold in M9 medium supplemented with

10 g/L of casamino acids, 2 g/L of glucose (BD, USA),

25 lg/mL of vitamin B1, 20 mM magnesium sulphate,

ampicillin (50 lg/mL) and chloramphenicol (10 lg/mL)

grown in 3 L Erlenmeyer flasks on an Infors shaker

(200 rpm) at 37 �C to an OD540 of 0.8–1.0, induced with

100 lg/mL of IPTG and cultivated for 4 h. Cells were

harvested by centrifugation.

To purify the mosaic AP205-am-DIII and AP205-op-

DIII VLPs, 3 g of wet, fresh cells were homogenised in

9 mL of lysis buffer containing 50 mM Tris–HCl (pH 8.0),

5 mM EDTA, 50 lg/mL PMSF and 0.1 % Triton X-100

and then ultrasonicated five times for 15 s each time at

22 kHz at 45 s intervals, while keeping the cells on ice.

After centrifugation at 10,000 rpm for 30 min, the super-

natant was loaded onto a Sepharose CL-2B column

(70 9 2 cm). NET buffer [0.15 M NaCl, 20 mM Tris–HCl

(pH 7.8), 5 mM EDTA] with 0.02 % Brij58 was used for

elution at a velocity of 2 mL/h, 90 min/3-mL fraction.

VLP-containing fractions were detected by native 0.8 %

Fig. 1 Schematic representation of the recombinant DIII constructions. The nucleotide and aa sequences and the locations of restriction sites at

the joining points are shown. Undefined aa residues are depicted by X

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agarose (TopVision LE GQ, Fermentas, Lithuania) gel

electrophoresis in 1xTAE buffer [40 mM Tris (pH 8.4),

20 mM acetic acid, 1 mM EDTA] (Fermentas). The VLP-

containing fractions (typically fractions 15–28) were

pooled and the material was loaded onto a Sephadex A50

(5–7 9 1 cm) column, which does not retard VLPs, to

remove nucleic acids. Unbound material containing VLPs

was washed out with NET buffer (without Brij58) under

spectrophotometric control. After concentration on an

Amicon Ultra-15 centrifugal filter device (MWCO 30,000;

Merck Millipore, USA), the samples were loaded onto a

Sepharose CL-4B column (48 9 1.5 cm) and eluted with

NET buffer at a velocity of 2 mL/h/fraction. The fractions

detected by native agarose gel electrophoresis (NAGE)

(typically fractions 17–25) were pooled, concentrated on

the Amicon device as described above, and subjected again

to gel filtration on the Sepharose CL-2B column

(60 9 1.5 cm) in NET buffer by collection of 2-mL frac-

tions. The VLP-containing fractions (typically fractions

24–33) were pooled, concentrated on the Amicon device to

3 mL, and loaded onto a pre-formed 5–36 % sucrose gra-

dient (sucrose concentration (w/w) layers: 36 % to 3 mL;

30 % to 3 mL; 25 % to 6 mL; 20 % to 8 mL; 15 % to

6 mL; 10 % to 6 mL; 5 % to 3 mL; in Polyallomer

25 9 89 mm tubes) for centrifugation in a Beckman

Coulter Optima L-100XP ultracentrifuge (rotor SW32 Ti)

at 20,500 rpm for 13 h at ?4 �C. Fractions of 1 mL were

collected from the pierced bottom of the tube. The VLPs

usually appeared around fraction 12, which was in the first

third from the bottom. Sucrose was removed by Amicon

concentrator to a volume of 1.5 mL in NET buffer and

1.5 mL of glycerol was added. VLP preparations with a

typical protein concentration of 8 mg/mL (31 OD260 units/

mL) were stored at -18 �C. The yield of VLPs reached

8 mg/g of wet cells.

To purify the GA-DIII derivative, the debris of the cell

lysate prepared as described above and centrifuged at

10,000 rpm for 30 min was eluted by 7 M urea in water

and loaded onto a DEAE cellulose column (1 9 5 cm) in

20 mM Tris–HCl (pH 8.6). The unbound material was

collected in fractions of 3 mL. The most pure fractions

were pooled, subjected to ammonium sulphate precipita-

tion at 50 % saturation, dissolved in 7 M urea and diluted

to 10 lg/mL in a 50 mM sodium carbonate buffer, pH 9.6,

for coating on ELISA plates.

Expression and Purification of the DIII Protein

Escherichia coli C2566 cells were transformed with the

pET28b-DIII plasmid (Fig. 1). Single colonies were sus-

pended in tubes containing 5 mL of LB medium with

50 lg/mL of ampicillin and grown without shaking at

37 �C for 16 h. The prepared inoculum was diluted tenfold

in 2TY medium containing 20 lg/mL of ampicillin in 2 L

flasks, incubated on an Infors shaker (200 rpm) at 37 �C to

an OD540 of 0.8–1.0, induced by adding IPTG to 100 lg/

mL and cultivated for 3.5–4.5 h. Cells were harvested by

centrifugation.

To purify the DIII protein, 1 g of cells was homogenised

and ultrasonicated in 4 mL of lysis buffer, and the super-

natant was discarded. The pellet was extracted with 4 mL

of 7 M urea in water. To remove the nucleic acids, two

alternative techniques were used: (i) proteins in the

supernatant were precipitated by adding ammonium sul-

phate to 50 % saturation for 20 h at 4 �C, the debris was

dissolved in 7 M urea and the ammonium sulphate pre-

cipitation was repeated and (ii) the supernatant was loaded

onto a DEAE cellulose column equilibrated with 20 mM

Tris–HCl (pH 8.6), and the unbound material was collected

and precipitated with ammonium sulphate at 50 % satura-

tion. The pellets obtained from both versions were washed

by water portions of 200 lL to remove any remaining

nucleic acids. After centrifugation at 10,000 rpm for

30 min, the samples were resuspended in a minimal vol-

ume of 7 M urea containing 5 mM dithiotreitol (Sigma-

Aldrich, USA), clarified by centrifugation at 6,000 rpm,

and refolded by dialysing the 3 mL sample for 3–4 days

against 4–5 changes of 50 mL of buffer containing 2 M

urea and 0.5 M arginine-HCl (pH 8.0) [41]. After clarify-

ing at 6,000 rpm for 15–20 min, the sample was subjected

to gel filtration on a Sepharose CL-4B column (30 9 1 cm)

and eluted in PBS buffer (0.01 M phosphate, pH 7.4,

0.138 M NaCl, 0.0027 M KCl) at a velocity of 1 mL/h,

90 min/1.5-mL fraction. The final samples were stored

frozen at -18 �C.

Detection of Protein and Nucleic Acids

All measurements were performed on Biochrom (Bio-

chrom Ltd., UK) and Nanodrop (Thermo Scientifics, USA)

spectrophotometers. The amount of protein (in the presence

of nucleic acids) was estimated according to [42]. The VLP

preparations were compared with respect to the presence of

protein and nucleic acids using NAGE and double radial

immunodiffusion (DRI) according to the method of

Ouchterlony using rabbit polyclonal anti-AP205 antibod-

ies. For NAGE and DRI, 1 and 0.8 % TopVision LE GQ

Agarose (Fermentas) in TBE buffer and in PBS buffer were

used, respectively, with subsequent Coomassie Blue R-250

(60 lg/mL of Coomassie Blue R-250 in 10 % acetic acid)

staining of the gels.

Protein samples were analysed on 15 % SDS-PAGE

gels with subsequent Coomassie staining. For Western

blots, the polyclonal rabbit anti-AP205 was used at a

1:1000 dilution. All chemicals were from Sigma-Aldrich.

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The ratio of DIII-containing versus initial AP205 pro-

teins was determined by analysis of Coomassie-stained gels

with the free ImageJ program (http://rsbweb.nih.gov/ij/).

Electron Microscopy and Dynamic Light Scattering

Analysis

For electron microscopy, VLPs in suspension were adsor-

bed to carbon-Formvar coated copper grids and negatively

stained with a 1 % uranyl acetate aqueous solution. The

grids were examined with a JEM-1230 electron microscope

(Jeol Ltd., Tokyo, Japan) at 100 kV.

The size of the particles was detected by dynamic light

scattering (DLS) in a Zetasizer Nano ZS (Malvern Instru-

ments Ltd, UK) instrument.

Immunogenicity of the DIII-Containing Proteins

Female BALB/c mice, 6–8 weeks of age and obtained from

the Latvian Experimental Animal Laboratory (Riga Stra-

dins University), were maintained at the Biomedical

Research and Study Centre under pathogen-free conditions.

The experiments were approved by the Latvian Animal

Protection Ethics Committee and the Latvian Food and

Veterinary service, permission No. 31/23.10.2010. Groups

of five mice were immunised sub-dermally on the back of

the animals with 25 lg of protein (diluted in 0.2 mL of

PBS) per mouse on days 0, 14 and 28. For immunisation

with adjuvants, 250 lg of Alum for all three immunisations

and 100, 250 and 500 lg of SiO2 for the first, second and

third immunisation, respectively, were used. To assess the

humoral response, the animals were bled on days 7, 14 and

28, reimmunised on days 15 and 29, and sacrificed on day

42. The anti-DIII titres in the sera were determined with a

direct ELISA using plates coated with DIII protein or GA-

DIII fusion protein. The anti-AP205 titres were determined

with a direct ELISA using plates coated with the AP205

VLPs.

For the direct ELISA, 96-well microplates (Nunc, USA)

were coated with the appropriate proteins using 100 lL of

protein solution (10 lg/mL in 50 mM sodium carbonate

buffer, pH 9.6) per well. The plates were incubated with

the protein solution overnight at 4 �C. After the plates were

blocked with 1 % BSA in PBS for 1 h at 37 �C, serial

dilutions of the sera were added to the wells, and the plates

were incubated at 37 �C for an additional 1 h. After

washing three times with PBS containing 0.05 % Tween-

20, 100 lL of horseradish peroxidase conjugated anti-

mouse antibody (Sigma-Aldrich) was added at a 1:10,000

dilution. After incubation at 37 �C for 1 h, the plates were

washed, and OPD substrate (Sigma-Aldrich) was added for

colour development. A Multiskan (Sweden) was used to

measure the absorbance at 492 nm. The end-point titres

were defined as the highest serum dilution that resulted in

an absorbance value three times greater than that of the

control sera obtained from unimmunised mice.

The IgM and IgG subsets in the sera of the immunised

mice were detected with isotype specific ELISAs using a

mouse monoclonal antibody isotyping reagent and an anti-

goat/sheep IgG peroxidase conjugate (Sigma-Aldrich).

For the competitive ELISA, 96-well microplates were

coated with the DIII protein using 100 lL of protein

solution (10 lg/mL) per well. After the plates had been

coated, 50 lL aliquots of serial dilutions of competing

protein AP205-am-DIII and 50 lL of the anti-DIII were

added to the wells simultaneously. The 1:800 dilution of

the anti-DIII with an OD492 value within the range of

0.5–0.6 in the control samples without competing protein

was used. After incubation at 37 �C for 1 h, the micro-

plates were processed as described above. The percent

inhibition (I%) of antibody binding by the competing

protein was calculated as follows:

I% = [(OD492 test sample - OD492 negative control)/

(OD492 positive control - OD492 negative control)] 9 100.

The molar amounts of the proteins necessary for 50 %

inhibition (I50) were calculated.

Results

Construction, Expression and Purification

of Recombinant AP205 Variants Carrying the DIII

Epitope

The construction of the recombinant AP205-am-DIII and

AP205-op-DIII genes, where the DIII sequence is C-ter-

minally fused to the AP205 CP over amber or opal trans-

lation termination codons, is depicted in Fig. 1. Direct

expression of the DIII sequence (Fig. 1) was performed to

have a source of the DIII for ELISA testing after immu-

nisation of mice with AP205 VLPs carrying the DIII

sequence. Furthermore, we used highly purified and

refolded DIII protein as a congruent for immunisation of

mice in parallel with the mosaic AP205-derived VLPs.

The expression level of both AP205-am-DIII and

AP205-op-DIII polypeptides, as determined from SDS-

PAGE of total SDS-mercaptoethanol cell lysates, corre-

sponded, in general, to the suppressor capacity of the

appropriate E. coli strains (Fig. 2a). Approximately 50 %

of the total target proteins appeared in the soluble cell

fraction, similar to the separation that occurs during

expression of non-chimeric AP205 or other VLP-producing

(RNA phage Qb, hepatitis B core antigen) genes (not

shown). The non-chimeric AP205 carrier did not demon-

strate any difference in solubility in comparison to the

AP205-DIII fusions. The ratio of AP205-DIII fusions to

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AP205 in the initial E. coli lysates was *50 %. During the

preparation of ultrasonicated cell lysates, a fraction of

AP205 dimers appeared and remained relatively stable

during boiling in Laemmli’s buffer; however, extensive

boiling of the final purified samples led to the disappear-

ance of the AP205 dimer band (Fig. 2b).

The mosaic VLPs yielded, on average, *8 mg of

purified AP205-DIII per gram of wet cells. After extensive

purification, the mosaic AP205-DIII VLPs demonstrated a

high purity of the target proteins (up to 90 %) with a ratio

of AP205-DIII to AP205 carrier of *0.12–0.17 to 1 within

the particles.

The minimal contamination of purified AP205-DIII

VLPs with host proteins (Fig. 2b) can be explained by

intra-particle occlusion of bacterial proteins rather than by

adherence on the VLP surface. A gradual loss of the chi-

meric AP205-DIII component of VLPs was observed dur-

ing the purification process. This gradual loss would

happen if VLPs enriched with chimeric DIII-harbouring

monomers are less stable than VLPs with a lower content

of chimeric monomers and are therefore preferentially

destroyed during purification.

Properties of Mosaic AP205 VLPs Carrying the DIII

Epitope

Double radial Ouchterlony immunoprecipitation, which

could be regarded as the most specific test for the existence

of the VLPs, demonstrated full confluence, without any

‘spurs’, of the precipitation lines formed by the AP205-am-

DIII and AP205-op-DIII VLPs with the precipitation line

produced by the non-chimeric AP205 VLPs (Fig. 2c, top).

Therefore, full ‘Ouchterlony identity’ of the AP205-

derived VLPs was demonstrated even though they differed

with respect to the presence of the AP205-DIII fusions.

Disruption of the VLPs by boiling in Laemmli’s buffer

prevented formation of the precipitation lines in the

Ouchterlony’s test (Fig. 2c, bottom).

According to electron microscopy analysis, purified

mosaic AP205-derived particles were indistinguishable

from non-purified items observed in the initial E. coli

lysates (not shown), as well as from the original AP205

carrier particles (Fig. 3).

Direct measurement of the particle size in solution using

the DLS method revealed particles with a close-to-expec-

ted diameter of 32.7 nm in the case of the control AP205,

but slightly differing diameters of 32.7 and 37.8 nm for

AP205-am-DIII and AP205-op-DIII, respectively (Fig. 4).

The DIII sequence appeared on the surface of the VLPs

because it was fully accessible to anti-DIII antibodies in a

competitive ELISA test (not shown).

The content of the encapsidated RNA within purified

mosaic AP205-DIII VLPs was estimated as 1,180 nucleo-

tides per particle; the estimated RNA content remained

constant after additional purification steps.

Double radial Ouchterlony immunodiffusion using

polyclonal rabbit anti-AP205 antibodies and NAGE con-

firmed stable association of the encapsidated RNA with the

VLPs (not shown). Therefore, we think that the residual

nucleic acids are not placed on the surface of mosaic par-

ticles, since nucleic acids are resistant to ribonuclease

treatment (not shown) and are not removed not only during

Ouchterlony immunodiffusion process, but also by DEAE

Sephadex chromatography, sucrose gradient centrifugation

or ammonium sulphate precipitation.

Properties of Regular AP205-DIII and GA-DIII

Fusions, and Purification of the Directly Expressed DIII

Protein

We were motivated to generate mosaic AP205-derived

particles because our early efforts to generate regular

Fig. 2 Monitoring of the expression and purification process of the

mosaic AP205-DIII VLP variants. a SDS-PAGE Western blot (with

polyclonal anti-AP205 antibody) of total protein in SDS-mercap-

toethanol lysed cell samples (T), supernatants of the ultrasonicated

cell lysates (S), and debris of the ultrasonicated cell lysates after

solubilisation in 7 M urea (D). b Coomassie stained SDS-PAGE of

the final purified preparations of the mosaic AP205-DIII particles,

c double radial Ouchterlony’s immunoprecipitation analysis of

AP205-DIII mosaics: native VLPs (top), VLPs boiled in Laemmli’s

buffer (bottom). The polyclonal anti-AP205 antibody was placed in

the central hole

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AP205-DIII and/or GA-DIII VLPs were unsuccessful. When

regularly fused AP205-DIII or GA-DIII genes without any

intervening suppressor codons (Fig. 1) were expressed,

chimeric target proteins appeared as insoluble products

without any signs of self-assembly in bacterial cells (not

shown). The attempts to refold them in vitro, also in the

presence of the initial AP205 as a helper, failed (not shown).

Direct expression of DIII protein led to a substantial

yield, 12 mg/g of cells, of insoluble recombinant product.

The DIII protein was purified and refolded by an arginine-

mediated renaturation procedure [41], as described in the

Materials and Methods. The final yield of the renatured

product after CL4B column chromatography reached 3 mg/

g of cells. The quality of the refolded DIII protein is shown

in Fig. 2b. The purified DIII protein did not form aggre-

gates in solution and DLS analysis demonstrated particles

with a radius of 4.8 nm (Fig. 4).

Immunisation of Mice with DIII-Carrying Proteins

In Fig. 5, we present immunisation data on the AP205-op-

DIII VLPs only because analogous data on the immuni-

sation of mice with the AP205-am-DIII VLPs demonstrate

general similarities with the presented data.

The antibody response in mice against the DIII epitope

was demonstrated by direct ELISA on two different anti-

gens coated onto solid support: (i) purified DIII protein and

(ii) purified regular GA-DIII fusion, where DIII was added

C-terminally to the CP of RNA phage GA. RNA phage GA

is not cross-reactive immunologically with RNA phage

AP205. Titration on differently coated solid supports led to

the conclusion that the fused GA-DIII protein was more

Fig. 3 Electron microscopy analysis of the purified mosaic AP205-DIII VLPs. a AP205-am-DIII, b AP205-op-DIII, c AP205 VLPs as a control.

Scale bar 50 nm

Fig. 4 The size of particles in the purified samples measured by DLS

analysis. The results of the DLS size distribution are shown with the

particle radius in nm on the x-axis and the number of particles in % on

the y-axis. The arrows indicate the radius of the particles

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sensitive (up to four times) as a support for anti-DIII anti-

body detection than the purified DIII polypeptide (Fig. 5a).

After immunisation of mice without any adjuvants, the

AP205-op-DIII VLPs induced an evident anti-DIII

response (Fig. 5a, top, slot 1); however, the response was

about four times weaker than the appropriate anti-AP205

carrier response (Fig. 5a, bottom, slot 1). Formulation of

the AP205-DIII VLPs in the Alhydrogel adjuvant prefer-

ably enhanced anti-DIII (Fig. 5a, top, slot 2). In all cases,

the maximal anti-DIII response was found after two boosts

on day 42 of immunisation (Fig. 5a, top).

The DIII polypeptide appears to be a weak immunogen

after immunisation of mice without any adjuvant (Fig. 5a,

top, slot 3). Formulation of the DIII polypeptide in Alhy-

drogel (Fig. 5a, top, slot 4) or silicon dioxide (Fig. 5A, top,

slot 5) enhanced the anti-DIII response to the level of the

anti-DIII response induced by the AP205-op-DIII VLPs in

Alhydrogel (Fig. 5a, top, slot 2).

Isotyping of the induced antibodies revealed a remark-

able increased ability of the AP205-op-DIII VLPs over the

DIII protein to induce anti-DIII antibodies of the IgG and,

specifically, the IgG2a isotype without any adjuvants

(Fig. 5b, top, slots 1 and 3). Without adjuvants, the DIII

polypeptide was unable to switch antibody production from

the IgM to the IgG isotype (Fig. 5b, top, slot 3). By im-

munising with adjuvants, the DIII protein acquired the

ability to perform this switch and induce IgG1 antibodies

(Fig. 5b, top, slots 4 and 5), whereas the AP205-op-DIII

VLPs demonstrated clear competence to induce anti-DIII

antibodies of both IgG1 and IgG2a/b isotypes (Fig. 5b, top,

slots 2, 4, and 5).

The AP205 carrier within the AP205-op-DIII VLPs

induced a broad spectrum of IgM, IgG1 and IgG2a/b

antibodies both in the absence and presence of the adjuvant

(Fig. 5b, bottom, slots 1 and 2).

Discussion

After the WNV-neutralising efficacy of AP205 VLPs

decorated by chemically coupled WNV DIII sequence was

shown [33], it seemed intriguing to achieve RNA phage

coat-driven VLPs carrying the DIII sequence by fusion of

the appropriate genes. Moreover, AP205-DIII fusions may

eliminate the potential uncertainty of the chemical cou-

pling [33], because the DIII sequence contains two internal

cysteine residues. Because self-assembly of the C-terminal

AP205-DIII and GA-DIII fusions in vivo and attempts to

refold them in vitro failed, construction of mosaic AP205

VLPs bearing the DIII epitope on the VLP surface was

performed in the present study.

The idea that the assembly non-competent VLP mono-

mers might be rescued into mixed or mosaic particles in the

presence of native VLP monomers as helpers was first

described for the hepatitis B virus (HBV) surface antigen

(HBsAg) [43]. By simultaneous expression of two genes,

mosaic HBsAg particles carrying poliovirus [44] and malaria

[45, 46] epitopes have been purified and successfully applied

as vaccine candidates. Interestingly, mosaic HBsAg-polio-

virus particles induced much higher titres of neutralising

antibodies to poliovirus than did the homogenous ones [44].

Additionally, mosaic V3:Ty-VLPs have been produced that

carry various V3 loops of different HIV isolates on the same

Ty particle [47]. Furthermore, incorporation of mutated VLP

Fig. 5 Immunogenicity of the DIII-carrying proteins. a Average

antibody titres to DIII (top) and AP205 carrier (bottom) for the sera

from five animals are shown. b Isotyping of the antibodies induced

against DIII (top) and AP205 carrier (bottom) after the immunisation

of mice with DIII-carrying proteins. Antibodies were diluted 1:50

with PBS

Mol Biotechnol

123

monomers into well characterised, highly symmetric icosa-

hedral VLPs has been described for the hepatitis B core

antigen (HBcAg) [48, 49]. In the case of HBcAg, the real

presence of both homo- and hetero-dimers within mosaic

particles has been documented [50].

A strategy for constructing mosaic particles was started by

introducing a linker containing translational stop codons

(UGA or UAG) between the sequences encoding a monomer

body and a foreign protein sequence, with subsequent

simultaneous synthesis of both the initial VLP monomer as a

helper moiety and a read-through fusion protein containing a

foreign sequence. This strategy was applied for RNA phage

Qb coats [51–53] and C-terminally truncated HBcAg for

exposition of hantavirus [54–56] and HBV preS [57] epitopes.

The same mosaic VLP strategy was used here in the case

of the RNA phage AP205 coats, which demonstrated def-

inite advantages as promising VLP carriers by construction

of putative prophylactic and therapeutic vaccine candidates

[36].

It is noteworthy that in the present study, both amber and

opal suppressions demonstrated similar outcomes of the

target proteins, a situation that is not always the case. For

example, the yields of analogous AP205 mosaics with Af-

fibody differed strongly between the amber and opal sup-

pression variants (Renhofa et al. personal communication).

One of the most prospective features of the mosaic

AP205 derivatives carrying the DIII epitope is their ability

to induce IgG2 antibodies, which is a result of Th1 pathway

activation. As is known for other VLP carriers, e.g. for

HBcAg VLPs, the Th1 priming and induction of IgG2

isotype antibodies correlates with the presence of encaps-

idated RNA [58, 59]. The similar Th1/Th2 switch con-

nected with the loss of encapsidated RNA has been

described for full-length and C-terminally truncated HBc

variants carrying HBV preS1 [60] and HCV [61] epitopes.

Encapsidated RNA functions in this case as a TLR-7 ligand

[62]. The cause of similar behaviour of AP205 mosaics

carrying the DIII epitope could be a subject of further

immunological studies.

Acknowledgments We wish to thank Dr. B. E. Martina for pro-

viding us with the pTrcHis2-WNV plasmid, Juris Ozols, Guntars

Zarins, Dace Priede, and Inara Akopjana for excellent technical

assistance. This work was supported by a Latvian grant 2010/0261/

2DP/2.1.1.1.0/10/APIA/VIAA/052 and FP7 Grant 261466 Vectorie.

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